ThermoFisher Scientific NanoDrop Eight, User Manual

Page: 53 / 152

5
Protein Applications
Measure Protein A280
Thermo Scientific NanoDrop Eight User Guide 53
Most sources report extinction coefficients
for proteins measured at or near 280 nm in
phosphate or other physiologic buffer.
These values provide sufficient accuracy for
routine assessments of protein
concentration. Published Extinction Coefficients
Published extinction coefficients for proteins may be reported
as:
• wavelength-dependent molar absorptivity (or extinction)
coefficient (

) with units of M
-1
cm
-1
• percent solution extinction coefficient (

1%) with units of
(g/100 mL)
-1
cm
-1
(i.e., 1% or 1 g/100 mL solution
measured in a 1 cm cuvette)
• protein absorbance values for 0.1% (i.e., 1 mg/mL)
solutions
Tip : Assess published values carefully to ensure unit of
measure is applied correctly.
The equation at the right shows the
relationship between molar extinction
coefficient (

molar
) and percent extinction
coefficient (

1%).
Conversions Between

molar
and

1%
(

molar
) * 10 = (

1%) * (MW
protein
)
Example: To determine percent solution extinction coefficient
(

1%) for a protein that has a molar extinction coefficient of
43,824 M
-1
cm
-1
and a molecular weight (MW) of 66,400
daltons (Da), rearrange and solve the above equation as
follows:

1% = (

molar
* 10) / (MW
protein
)

1% = (43,824 * 10) / 66,400 Da)

1% = 6.6 g/100 mL
To determine concentration (c) of a sample
in mg/mL, use the equation at the right and
a conversion factor of 10.
Tip : The NanoDrop Eight software
includes the conversion factor when
reporting protein concentrations.
Conversions Between g/100 mL and mg/mL
C
protein
in mg/mL = (A /

1%) * 10
Example: If measured absorbance for a protein sample at
280 nm relative to the reference is 5.8 A, protein
concentration can be calculated as:
C
protein
= (A /

1%) * 10
C
protein
= (5.8/6.6 g/100 mL) * 10
C
protein
= 8.79 mg/mL